CENTRAL NERVOUS SYSTEM REGULATION OFINTESTINAL MOTILITY: ROLE OF ENDOGENOUS

OPIOID PEPTIDES (ENDORPHINS, ENKEPHALINS)

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Authors GALLIGAN, JAMES JOSEPH.

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Galligan, James Joseph

CENTRAL NERVOUS SYSTEM REGULATION OF INTESTINAL MOTILITY: ROLE OF ENDOGENOUS OPIOID PEPTIDES

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CENTRAL NERVOUS SYSTEM REGULATION OF INTESTINAL MOTILITY:

ROLE OF ENDOGENOUS OPIOID PEPTIDES

by

James J. Galligan

A Dissertation Submitted to the Faculty of the

PROGRAM IN PHARMACOLOGY AND TOXICOLOGY

In Partial Fulfillment of the Requirements

For the degree of

DOCTOR OF PHILOSOPHY

In the Graduate College

THE UNIVERSITY OF ARIZONA

1 983

THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE

As members of the Final Examir,9.tion Committee, we certify that we have read

the dissertation prepared by James J. Gallisan

entitled _____ C_en __ tr_a_1 __ N_e_r_v_o_u_s __ S~y_s_t_e_m __ R_e~g_u_l_a_ti_o_n __ o_f_·_I_n_t_e_s_t_i_n_a_l __ M_o~t_i~l~i~ty~: ____ _

Role of Endogenous Opioid Peptides.

and recommend that it be accepted as fulfilling the dissertation requirement

for the Degree of Doctor of Philosophy ----------------.----~~--------------------------------

Date

Date

Date

Date

Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the disser.tation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my dire~~tion and recommend tha,t it be accepted as fl1lfilling the dissertation

Date J

STA'rEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillemnt of the requirments for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under the rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of the source is made. Rpquests for permission for extended quotation from of reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College wlllm in his judgement the proposed use of the material in in the interests of scholarship. In all other instances, however, per-mission must be obtai ned from th(~ author.

DEDICATION

This dissertation and all the work involved in its completion is dedicated to my father.

iii

ACKNOWLEDGMENTS

A special thanks to Dr. David L. Kreulen who provided expert assistance and advice on many of these experiments and also allowed the use of his laboratory during the ~n v~tro studies.

iv

TABLE OF CONTENTS

LIST OF ILLUSTRATIONS •••••• CI •••••••••••••••••••••••••••••••

LIST OF TABLES ............................................. ABSTRACT ...................................................

INTRODUCTION •••••••••••••••• 0 ••••••••••••••••••••••••••••••••••

Page vii

x

xi

1

Hormonal Control of Intestinal Motility.................... 2 Intrinsic Neural Control of Intestinal Motlity ••••••••••••• 3 Extrinsic Neural Control of Intestinal Motility............ 5 Patterns of Intestinal Motility............................ 7 Opiates and Motility....................................... 11 Irritable Bowel Syndrome ••••••••••••••••••••••••••••••••••• 18 Statement of Problem •••••• ~................................ 22

METHODS ........................................................ 24 Surgical Preparation of Animals for Intestinal Transit Studies •••••••••••••••••••••••••••••••••••••••••••• 24

Intracerebroventricular Cannulas •••••••••••••••••••••• 26 Hypophys~ctomy •••••••••••••••••••••••••••••••••••••••• 27 Spinal Cord Section •••••••••••••••••••••••••••••••••• 27 Subdiaphramatic Vagotmy ••••••••••••••••••••••••••••••• 27

Evaluation of Intestinal Transit and Gastric Emptying •••••• 28 Gastric Emptying ••••••••••• ~.......................... 28 Small and Large Intestinal Transit •••••••••••••••••••• 29

Effects of Morphine on Small Intestinal Transit and Gastric Emptying ••••••••••••••••••••••••••••••••••••••• 31 Direct Measurement of Small Intestinal Motility............ 31 Effects of Opioid Peptides on Small Intestinal Transit in Castor Oil-Treated Rats ••••••••••••••••••••••••• 34 Effects of Electroconvulsive Shock on Gastrointestinal Motility and Analgesia ••••••••••••••••••••••••••••••••••••• 35 Effects of Inescapable Footshock on Gastrointestinal Motility and Analgesia ••••••••••••••••••••••••••••••••••••• 36 Kyotorphin Effects on Intestinal Transit and Analgesia ••••• 37 Determination of Opioid Receptor Selectivity of Agonists in Vitro ••••••••••••••••••••••••••••••••••••••• 38 Determination of the Opioid Receptors Mediating the Analgesic and Intestinal Motility Effects of Centrally-Administered Opioids ••••••••••••••••••••••••••••• 41

v

TABLE OF CONTENTS continued

Page

RESULTS 43

Effects of Morphine on Gastric Emptying and Small Intestinal Transit and Motility............................ 43 Effects of Opioid Peptides on Intestinal Transit in Castor Oil-Treated Rats ••••••••••••••••••••••••• 52 Effects of ECS on Gastrointestinal Motility and Analges 1a .•..•••...•............••••.•....•...•.•...••. 64 Effects of IFS on Gastrointestinal Motility and Analgesia •••••••••••••••••••••••••••••••••••••••••••••• 67 Effects of Kyotorphin of Small Intestinal Transit a nd Analgesia •••••••••••.•••.••••.••••••••••••....•.••..•.• 67 In Vitro Determination of Receptor Selectivity............. 67 Effects of Receptor Selective Agonists on Small Intestinal Transit and AnalgeSia •••••••••••••••••••••••••••••••••••••• 75 Relative Potencies In Vivo ••••••••••••••••••••••••••••••••• 84

DISCUSSION 88

REFERENCES 110

vi

LIST OF ILLUSTRATIONS

Figure

1. Schematic drawing of the implant used in these studies to record intestinal motility from

Page

unanesthetized rats ••••••••••••••••••••••••••••••••• 33

2. Distribution of radiochromium in the small intestine of rats treated with intracerebro-ventricular morphine or saline ••••••••••••••••••••••• 44

3. Dose-response curves for morphine induced-inhibit-ion intestinal transit in fasted rats •••••••••••••••• 46

4. Inhibition of gastric emptying of a radioactive marker by morphine in fasted rats ••••••••••••••••••• 48

5. Typical recording of duodenal motility on the unanesthetized rat before and after morphine treatment ..•.••..••..•.•....•.••..•••.••••..••••••..• 49

6. Dose-response curves for inhibition of intestinal motility by morphine given i.c.v. or s.c. to unanesthetized rats •••••••••••••••••••••••••••••••••• 51

7. Inhibition of intestinal transit in castor Oil-pre-treated rats by a-endorphin and DALA and antagonism by naloxone •..................••..••.••........••..•. 55

8. Inhibition of intestinal transit in castor oil-pre-treated rats by a-endorphin and DALA and antagon-ism by naloxone ••••••••••••••••••••••••••••••••••• e.. 56

9. Effect of DANM-pretreatment on the antitransit actions a-endorphin, DALA and loperamide ••••••••••••••••••••• 57

10. Failure of vagotomy to alter the antitransit effects of i.e.v. a-endorphin •••••••••••.•••••••••••••••••••• 59

11. Failure of vagotomy to alter the antitransit effects of i.e.v. DALA ••••••••••••••••••••••••••••••••••••••• 60

12. Failure of spinal cord section to alter the antitran-sit effects of i.c.v. a-endorphin •••••••••••••••••••• 61

vii

LIST OF ILLUSTRATIONS-Continued

Figure Page

13. Failure of spinal cord section to alter the antitransit effects of i.e.v. DALA •••••••••••••••••••••••••••••••• 62

14. Failure of hypophysectomy to alter the antitransit effects of i.c.v. DALA or a-endorphin ••••••••••••••••• 63

15. Increase in hot-plate response times by rats treated with electroconvulsive shock (ECS) and antagonism by naloxone •••••••••••••••••••••••••••••••• 66

16. Increase in hot-plate response times by rats treated with inescapable footshock (Shock) and antagonism by naloxone ......•..•.............•. ~ . . • • . . . . . . . . . . . . . 69

17. Failure of kyotorphin to affect intestinal transit following intracerebroventricular administration •••••• 70

18. Time course of kyotorphin-induced increases in hot-plate latencies following intracerebroven-tricular administration of kyotorphin ••••••••••••••••• 71

19. Dose response curve for kyotorphin-induced analgesia in and antagonism by naloxone •••••••••••••••••••••••••••• 72

20. Inhibition of intestinal transit by DAGO and morphine •.•••••••••••••••••••••••••••••••••.•.•..•.•.• 76

21. Inhibition of intestinal transit by DALA and a-endorphin .•......•.....•..•.•..•••.....•....•...•••. 77

22. Inhibition of intestinal transit by DADL and DPLCE 78

23. Failure of DPLPE and DPDPE to affect intestinal transit ............................................... 79

24. Failure of U-50,488H to affect intestinal transit 80

25. Time course of analgesia produced by a-endorphin, DADL, DALA and morphine ••••••••••••••••••••••••••••••• 81

viii

LIST OF ILLUSTRATIONS-Continued

Figure Page

26. Time course of analgesia produced by DPLCE, DPLPE, DPDPE and DAGO ••••••••••••••••••••••••••••••••••••••• 82

27. Dose-response curves for analgesia produced by DPDPE, DPLPE, DPLCE and DAGO and antagonism by naloxone 83

28. Correlation of delta receptor selectivity with increases in analgesic EDSO and the EDSO for inhibition of small intestinal transit (S.I.T.)

ix

87

LIST OF TABLES

Table Page

1. Subcutaneuous naloxone antagonism of the small intestinal antitransit effects of subcutaneous or intracerebro-ventricular morphine •••••••••••••••••••••••••••••••••••••••• 47

2. Frequency of contractions in two areas of the small intestine of unanesthetized rats treated with intracerebroventricu1ar of subcutaneous morphine

3. Effects of opioid peptides on intestinal transit in

50

castor oil-treated rats ••••••••••••••••••••••••••••••••••••• 53

4. Effects of opioid peptides given i.c.v. to castor oil treated rats in small intestinal weight and body weight 10s8 •••••••••••••••••••••••••••••••••••••••••••• 58

5. Percent gastric emptying and geometric centers for small and large intestinal transit in sham and ECS treated rats ••••••••••••••• ~ •••••••••••••••••••••••••••• 65

6. Percent gastric emptying and geometric centers for small and large intestinal transit in IFS and sham treated rats .•...•........•..........•....•.•.•.•........... 68

7. Inhibition of the electrically-induced contractions of the G.P.I. and M.V.D. by normorphine and several opioid peptides ••••••••••••••••••••••••••••••••••••••••••••• 74

8. ED50 values for opioid-induced inhibition of ,small intestinal transit (S.I.T.) and for producing analgesia •••••••••••••••• 86

x

ABSTRACT

The complex interaction between the central nervous system, the

enteric nervous system and local and endocrine hormones enables drugs

affecting gastrointestinal motility to produce their effects through

multiple sites and mechanisms of action. Opiates are one class of

drugs which can have dramatic effects on gastrointestinal function and

the mechanisms for these actions have been the subject of intense study

in recent years. These changes in motility have assumed increased

importance following the discovery of several endogenous opioid pep-

tides.

In the present studies, centrally-administered morphine was more

potent than peripherally-administered morphine at inhibiting intestinal

propulsion and gastric emptying in rats. Direct measurment of intesti-

nal motility revealed that the antipropulsive effects of morphine were

due, to an inhibition of intestinal contractions.

The opioid peptide, a-endorphin, and a stabilized enkephalin

analog, [D-Ala2 , Met5]enkephalinamide, also inhibited intestinal pro-

pulsion only after central adminstration. These effects were not

blocked by a peripherally selective opioid receptor antagonist,

diallylnormorphinium.

These data indicated that there is an opioid sensitive mechanism

in the brain of rats that, when activated, can inhibit intestinal moti-

lity. Physiological activation, by electroconvulsive shock or inesca-

xi

pable footshock, or pharamcological activation by kyotorphin (Tyr-Arg)

treatment, did not affect gastrointestinal motility but did produce

naloxone-reversible analgesia. These data indicate that the opioid

mechanisms mediating analgesia and inhibition of intestinal motility

are independent and may be a function of different receptor systems.

Several opioid receptor selective agonists were used to deter-

mine the specific receptors mediating the analgesic and motility

effects of centrally-administered opioids. Mu selective agonists pro-

duced analgesia and inhibition of intestinal transit, while delta

receptor agonists produced'analgesia only. Kappa agonists did not pro-

duce analgesia or an inhibition of intestinal motility. Mu receptors

mediate the ~nAlgesic and intestinal motility effects of exogenously

administered opioids, while delta receptors can mediate analgesia with

out altering gut motility. It appears then, that electroconvulsive

shock, inescapable footshock and kyotorphin may produce their analgesic

effects by releasing enkephalins, which are delta selective agonists.

This accounts for the failure of these treatments to alter gastroin-

testinal motility while still producing the analgesic effects reported

here.

xii

INTRODUCTION

Control of gastrointestinal motility is a complex process and

has been the subject of intense study for over 100 years. The prin-

cipal consequence of the many controlling factors of gastrointestinal

motility is to coordinate contractions of the esophagus, stomach, small

and large intestines and the intervening sphincters so that food can be

digested, nutrients absorbed and waste excreted in an orderly and effi-

cent manner. While the fundamental basis for this process is simply an

inhibition or stimulation of contractions by gut smooth muscle, the

stimuli producing each of these effects can differ markedly. The pri-

mary concern of this discussion is the neural influences on small

intestinal motility and the control of contractions in this portion of

the gastrointestinal tract.

The motor functions of the mammalian small intestine are under

the control of intrinsic and extrinsic neural and hormonal influences.

The intrinsic nerves are those whose cell bodies reside in the enteric

nervous system, originally described by Langley (1921) as one of three

divisions of the autonomic nervous system. Extrinsic innervation con-

sists of those nerves whose cell bodies reside in the central nervous

system or in the prevertebral ganglia and form synaptic connections

with the intrinsic nervous system or gut smooth muscle directly.

1

2

Hormonal Control of Intestinal Moti~

Hormonal control involves both endocrine hormones and local or

paracrine hormones, both of which can influence gastrointestinal moti-

lity. Endocrine hormones are those substances which gain access to

their site of action only after release into the systemic circulation

by the hormone producing cell. In many cases, the target tissue is far

removed from the source of the hormone. Paracrine hormones are

released into the interstitial fluid by the hormone-producing cell and

generally affect only a few surrounding cells. Paracrine hormones are

locally acting substances. It is generally difficult to to make abso-

lute distinctions between paracrine and endocrine effects as many of

these substances can serve both functions. For example, bombesin and

somatostatin appear to have both endocrine and paracrine functions

(Solcia et al., 1981). To complicate this issue further, many of the

endocrine/paracrine substances are also found in the intrinsic and

extrinsic innervation of the small intestine. Somatostatin, chole-

cystokinin, substance P, serotonin and neurotensin are some of the

substances that are found in the central nervous system, the enteric

nervous system and in the endocrine/paracrine cells of the gut (Walsh,

1981; Solcia et al., 1981; Furness and Costa, 1982). Each of these

hormone/neurotransmitter substances can have dramatic effects on

intestinal motility in vivo, although it is difficult to determine if

these effects are neurally mediated (either centrally or peripherally),

hormonally mediated or both. Other substances such as secretin,

gastrin and glucagon appear to be located exclusively in endocrine

3

cells of the gastrointestinal tract (Solcia et al., 1981; Walsh, 1981).

This has been a brief survey of the types of hormonal influences that

exist which can influence intestinal motility. The remainder of this

review will focus on the intrinsic and extrinsic neural control of

intestinal motility.

Intrinsic Neural Control of Intestinal Motility

The intrinsic innervation of the mammalian small intestine con-

sists of those neurons whose cell bodies reside in one of the

ganglionated plexi located in the different layers of the gut wall.

The myenteric plexus (Aurebach's plexus) consists of very large ganglia

and an interconnecting network of nerve bundles which lies between the

longitudinal and circular muscle layers. The submucosal plexus

(Meissner's plexus) is made of smaller but more numerous ganglia and a

much finer interconnecting system of nerve fibers. The submucosal

plexus resides in the connective tissue of the submucosal layer

(Gabella, 1979; Gershon, 1981; Furness and Costa, 1980). There are

other nerve bundles and ganglia found in the small intestine, however,

the prominence and fine structure of these plexi seem to vary from spe-

cies to species. In all species, however, the myenteric and submucosal

plexi are the principal mediators of intestinal motility and intestinal

reflexes.

There is an abundance of peptide and non-peptide substances that

may be neurotransmitters in the enteric nervous system (Schultzberg et

al., 1980; Furness and Costa, 1980; Furness and Costa, 1982), however,

acetycholine may be the final common excitatory substance, while the

4

enteric inhibitory transmitter may be the final common inhibitory

substance. Acetycholine is present in the enteric nervous system in

higher concentrations than any other neurotransmitter substance

(Furness and Costa, 1982) and it is found in both intrinsic neurons

which innervate the muscle layers as well as in the interneurons within

the enteric ganglia. Stimulation of these enteric neurons releases

acetylcholine (Paton, 1957; Szerb, 1976) which produces a contraction

of intestinal smooth muscle. The direct action of acetylcholine on

smooth muscle is blocked by muscarinic cholinergic antagonists, while

the effects of acetylcholine released from enteric interneurons or from

extrinsic parasympathetic neurons are blocked by nicotinic cholinergic

antagonists (Kosterlitz and Lees, 1964; Furness and Costa, 1980).

The enteric inhibitory neurons are present in all areas of the

small intestine with the cell bodies located principally in the myen-

teric ganglia. The inhibitory neurons appear to be involved in local

intestinal relaxation as well as the in descending wave of inhibition

that is part of the peristaltic reflex (Costa and Furness, 1982).

Unfortunately, the nature of this non-cholinergic, non-adrenergic inhi-

bitory transmitter is unknown at this time. A large volume of evidence

has accumulated that indicates that this transmitter may be ATP or a

related purine nucleotide (Burnstock, 1972; Burnstock, 1978). However,

recent studies have provided direct evidence against a purine

nucleotide being the enteric inhibitory transmitter (Westfall, et al.,

1982; Bauer and Kuriyama, 1982). Vasoactive intestinal polypeptide has

also received some consideration as being this inhibitory substance

5

(Farhrenkrug et al., 1978; Furness and Costa, 1978) but more recent

data indicate that this peptide is not identical to the substance pro-

ducing intestinal relaxation following stimulation of the non-

cholinergic, non-adrenergic inhibitory neuron (Mackenzie and Burnstock,

1980; Bauer and Kuriyama, 1982). Thus, the identity of the inhibitory

neurotransmitter remains to be established.

Extrinsic Neural Control of Intestinal Motility

Extrinsic control of intestinal motility is mediated by the

parasympathetic and sympathetic divisions of the autonomic nervous

system. Parasympathetic innervation of the small intestine is derived

from the vagus nerve which contains both sensory afferents and motor

efferent fibers. The vagal motor efferents originate primarily in the

dorsal motor nucleus of the vagus located in the brain stem (Sato et

al., 1978) and synapse on intrinsic enteric neurons (Baumgarten, 1982).

Bayliss and Starling (1899) first reported that stimualtion of vagal

fibers can stimulate intestinal contractions followed by an inhibition

of motility. The excitatory response was blocked by atropine while the

inhibitory response was not affected by cholinergic or adrenergic anta-

gonists. These observations, later confirmed by many others, indicate

that vagal stimulation excites both the cholinergic post-ganglionic

neurons and the enteric inhibitory neurons (Roman and Gonella, 1981).

Sympathetic innervation of the small intestine consists of a

cholinergic preganglionic neuron which originates in the thoracic spi-

nal cord and a nor adrenergic neuron whose cell body is located in one

6

of the prevertebral ganglia (Furness and Costa, 1974; Baumgarten,

1982). The cholinergic preganglioinic fibe~s leave th~ spinal cord as

the splanchnic nerves and synapse with the postganglionic adrenergic

nerves located in the prevertebral ganglia (Norberg and Hamberger,

1964). Thes~ norepinephrine-containing cell bodies were identified

with flourescence-histochemical techniques and similar methods were

used to identify adrenergic nerve fibers in the intestinal wall

(Norberg, 1964; Jacobowitz,1965). These same investigators also noted

that no adrenergic cell bodies were present in the gut wall. The adre-

nergic fibers innervate the myenteric and submucosal plexi and their

terminals are generally found along the edges of the ganglia (Gabella,

1979; Manber and Gershon, 1979; Llewellyn-Smith et al., 1981). A few

adrenergic fibers also terminate in the circular and longitudinal

muscle layers (Wikberg, 1977).

Stimulation of the sympathetic nerves generally inhibits

intestinal contractions, while severing these nerves results in hyper-

motility. The norepinephrine released from sympathetic nerves can

affect smooth muscle directly or can alter intrinsic nervous activity

by inhibiting acetylcholine release from the intrinsic neurons. The

electrophysiological (Nishi and North, 1973; Hirst and McKirdy, 1974)

and the ultrastuctural evidence (Llewellyn-Smith et al., 1981) suggest

that the adrenergic receptors are located on cholinergic nerve ter-

minals. A more recent study has provided direct evidence for the pre-

sence of alpha2 adrenergic receptors on cholinergic neurons of the

myenteric plexus (Wikberg and Lefkowitz, 1982). Norepinephrine can

also relax smooth muscle directly by an action at both alpha and beta

adrenergic receptors. Alpha mediated inhibition is a result of an

increase in potasium and chloride conductance which hyperpolarizes the

cholinergic neuron (Bulbring and Tomita, 1969). The relaxation pro-

duced by beta receptor stimulation is preceeded by an intracellular

accumulation of cAMP (Anderson and Mohme-Lundholm, 1970).

Patterns of Intestinal Motility

7

The small intestine of many species generally exhibits two pat-

terns of motility (Weisbrodt, 1981). The fed pattern is difficult to

characterize and can depend on the type and quantity of food present in

the intestinal lumen. The fed pattern at a single intestinal location

consists of sequential contractions occurring at intervals of less than

one minute. These probably serve a mixing function as originally

described by Cannon (1902). These phasic contractions may be superim-

posed on tonic increases in intraluminal pressure as part of a pro-

pulsive peristaltic contraction which travels only short distances in

the fed state. The peristaltic contractions are a result of the

peristaltic reflex or law of the intestine (Bayliss and Starling

1899) that is a fundamental principle of intestinal motility. The

peristaltic reflex consists of a descending wave of inhibition below a

point of intestinal distention that is followed by an aborally moving

ring of intestinal contraction that originates above the point of

distention. The peristaltic reflex appears to be mediated solely by

intrinsic intestinal neurons (Costa and Furness, 1982). Myoelectric

recordings obtained from the small intestine of fed animals show an

almost random pattern of electrical spiking activity (Weisbodt, 1981).

8

In contrast, the fasted pattern of motility shows a regular

cyclic change in myoelectric activity. The fasted pattern of intesti-

nal electric activity, originally described by Szurszewski (1969), is a

regular change in myoelectric activity that occurs in cycles along the

entire length of the intestine. This migrating myoelectric complex

(MMC) is characterized by three distinct phases in dogs and humans

(Code and Mart1ett, 1975; Vantrappen et al., 1977). Phase 1 is a period

of relative quiet that is followed by Phase 2 or a period of random

electrical activity. Phase 3 is the most striking feature of the MMC

and is easily recognized as a period of intense and regular spiking

activity that may originate in the stomach or duodenum and migrates

abora11y. As electrical spiking activity is generally associated with

circular muscle contractions, Phase 3 is an abora11y moving ring of

intestinal contraction (Szurszewski, 1981). Code and Mart1ett (1975)

have also described a Phase 4 as a period of declining activity but

the existence of Phase 4 is not universally agreed upon. In fact, the

intense spiking of Phase 3 generally ceases abruptly. In addition to

humans and dogs, the MMC is seen in other species including sheep and

rabbits (Grive1 and Ruckebusch, 1972), pigs (Bueno et al., 1982) and

rats (Ruckebusch and Fioramonti, 1975). An understanding of the fac-

tors responsible for the MMC is important as much of the work con-

cerning neuronal control of intestinal motility has used the MMC as a

substrate for study. In addition, most of the studies dealing with

drug-induced changes in motility have also been carried out in fasted

animals.

9

Initiation of the MMC in the proximal small intestine appears to

be under hormonal control and there is substantial evidence that moti-

lin may be the responsible hormone. Motilin is a 22 amino acid peptide

isolated from hog intestine that was found to stimulate gastric moti-

lity (Brown et al., 1972). This hormone is found in highest con-

centrations in mucosal cells of the upper intestine (Walsh, 1981).

Intravenous administration of motilin to dogs or humans can initiate

premature MMC's in fasted subjects while the same treatment does not

affect the fed pattern of intestinal motility (Itoh, et al., 1978;

Ormsbee and Mir, 1978; Wingate et al., 1976). Plasma levels of motilin

also show a cyclic variation with peak concentrations occurring just

prior to or during Phase 3 activity (Itoh et al., 1979; Lee et al.,

1978) and stimuli which release endogenous motilin also initiate Phase

3 activity (Lee et al., 1978). Finally, treatment of fasted dogs with

an antibody to motilin can inhibit the formation of MMC's (Lee et al.,

1982). Thus there is substantial evidence that intiation of the MMC is

under hormonal control, however the role of intrinsic and extrinsic

nerves in the orderly propagation of the MMC is less clear.

The extrinsic innervation of the small intestine mayor may not

be important for the normal propagation of the MMC. Studies using iso-

lated loops of small intestine with .the extrinsic innervation intact

have shown that the MMC will frequently pass from the intestine to the

loop and back to the intestine in a normal fashion (Carlson et al.,

1972; Grivel and Ruckebusch, 1972). In addition, when the extrinsic

innervation of the isolated loop is removed the MMC will not appear in

the loop (Weisbrodt et al., 1975a). When a segment of intestine is

10

denervated extrinsically with the intrinsic nerves intact the MMC will

pass through the segment but at a much reduced velocity (Bueno et al.,

1979). These investigators also noted that when an isolated loop of

intestine was prepared and the remaining intestine reanastomosed, MMC's

would move from the intestine to the loop and back to the intestine at

some distance aboral to the anastomosis and after a considerable delay.

It was also noted that the number of complexes distal to the anastomo-

sis was greater than the number occurring on the proximal side.

Subsequent studies performed on isolate.d and denervated loops of

intestine in pigs have demonstrated that MMC's could be initiated and

migrate aborally in. the absence of extrinsic input (Aeberhard et al.,

1980; Itoh et al~, 1981). This effect was shown to be dependent on

intrinsic cholinergic neurons (Sarna et al., 1981). These data indi-

cate that the mechanism for initiation and propagation of the MMC is

intrinsic to the intestine but that extrinsic innervation may serve to

facilitate migration of the complex in an aboral direction.

It is clear then that normal functioning of the small intestine

appears to be under the direct control of the enteric nervous system

and that the extrinsic innervation can modify the contractile funct-

tions of the gut. The extrinsic innervation of the small intestine may

also serve to integrate digestive function with the other ongoing beha-

vioral and visceral processes of the animal. It should also beempha-

sized that while the enteric nervous system is relatively autonomous

many of the intestinal reflexes may require intact connections to the

prevertebral ganglia (Szursweski and Weems, 19776; Kreulen and

Szursweski, 1979). In additi~n, there are many excitatory and inhibi-

tory substances found in both the intrinsic nervous system and in the

extrinsic nerves (Furness and Costa, 1980; Furness and Costa, 1982;

Dalsgaard et al., 1983) all of which serve to modify the action of the

cholinergic excitatory neurons and the enteric inhibitory neurons of

the intrinsic nervous system.

Opiates and Intestinal Motility

11

The complex interaction between the central nervous system, the

intervening ganglia, the enteric nervous system and the multitude of

possible neurotransmitter substances has produced many sites and mecha-

nisms of action for drugs to affect intestinal motility. One class of

compounds which has been known for centuries to produce striking

changes in gastrointestinal function is the opiates.

Opium alkaloids have been used for many centuries for the

control of dysentery and other diarrheas and the mechanism of this

antidiarrheal effect has been the subject of considerable investigation

for 80-90 years. The reasons for such intensive study are that the

opiates can produce their intestinal effects through several mecha-

nisms and these effects can vary from species to species and from one

type of preparation to another. There is also an apparent paradox

seen in many species in that the well known constipating effects of

opiates are associated with increases in gastrointestinal motility.

Finally, the recent discovery of the endogenous opioid peptides has

led to the possibility that some disorders in gastrointestinal moti-

lity may be attributed to changes in opioid peptide levels or in acti-

vity of opioid containing neurons.

12

The early experiments dealing with the effects of morphine on

gastrointestinal motility have been reviewed by Plant and Miller

(1926). Many of the early investigators noted that subcutaneous

administration of morphine would often provoke spontaneous intestinal

contractions and would increase the irritability of the intestine when

provoked by certain stimuli. Other investigators noted that morphine

would delay the passage of food through the intestine. A principal

criticism of these early experiments was the use of a general anesthe-

tic during the experiment which was known, even at that time, to

suppress normal intestinal contractions and reflexes. Plant and Miller

(1926), using unanesthetized dogs, found that morphine would produce

dose-related increases in the frequency of phasic contractions as well

as tonic inc~eases in intraluminal pressure. These investigators also

noted qualitatively similar effects in human subjects after morphine

treatment. Following thes~ intitial observations, many other studies

showed that morphine could stimulate intestinal contractions in una-

nesthetized animals yet still produce constipation (Vaughan Williams,

1954).

This contradiction was resolved following experiments using an

isolated loop of intestine attatched at either end to a column of

water. Small doses of morphine increased contractile activity of

the segment yet propulsive work was reduced (Vaughan Williams and

Streeten, 1950). Morphine also increased the tone of the segment which

increased resistance and reduced the flow of intraluminal contents.

This observation has been supported by many subsequent studies which

indicate that morphine stimualtes a non-propulsive type of intestinal

motility and that the intestinal lumen becomes smaller reducing the

flow of the intraluminal contents (Bass and Wiley, 1965; Bass et al.,

1973).

The work described to this point had been performed using

13

intact animals and little information was provided concerning the site

of action. A number of isolated intestinal preparations have been used

to study the direct effects of opiates on intestinal motility. The

Trendelenburg preparation (Trendelenburg, 1907) and several modifica-

tions have been used to study the effects of opiates on the peristaltic

reflex. Briefly, a small piece of guinea-pig ileum is suspended in a

tissue bath with one end of the segment attatched to a tube which is

connected to a buffer resevoir. The ileal segment can be distended by

raising or lowering the level of the resevoir. Distention of the

segment produces a contraction of the longitudinal muscle followed by

progressive rings of circular muscle contraction and relaxation of the

longitudinal muscle. This reflex is mediated, at least partially, by

acetylcholine as hyoscine, atropine and hexamethonium will block the

contractile response (Kosterlitz and Lees, 1964). Morphine and other

opiates will also depress this reflex and their potency and efficacy

were closely correlated with their analgesic effects (Green, 1959;

Gyang et al., 1964). It is also interesting to note that this effect

was stereospecific as only levorotatory isomers of the opiates were

effective (Gyang et al., 1964). This action was not due to a depress-

ant effect on the smooth muscle as the preparation would respond norm-

ally to exogenous acetylcholine. 'Instead, the effects of opiates on

this reflex may be attributed to inhibition of acetylcholine release

from int'dnsic neurons (Schauman, 1957).

14

Low frequency electrical stimulation of segments of guinea-pig

ileum or the of longitudinal muscle-myenteric plexus preparation also

produces contractions that are inhibited stereospecifically by opiates.

This effect is also a result of an inhibition of acetylcholine release

from the intrinsic nerves of the ileal tissue (Paton, 1957).

Subsequent studies have demonstrated that the depressant effects of

morphine on the electrically-induced contraction are blocked by the

opiate receptor antagonist, naloxone (Kosterlitz and Watt, 1968).

Much of the work concerning the effects of opiates on intesti-

nal contractions and reflexes in vitro has been done using strips of

guinea-pig small intestine. The responses seen in these preparations

are generally an inhibition of contractions or of reflex activity.

However, as pointed out previously, studies in intact animals have

shown that morphine stimulates intestinal contractions. This is also

true of the dog-isolated intestine as intraarterial injections of

morphi~e produce both phasic and tonic contractions (Burks and Long,

1967). These investigators also reported a release of serotonin from

the intestinal segment following morphine treatment and that the

contractile response produced by morphine could be reduced with anta-

gonists of serotonin (Burks, 1973). These observations serve to

illustrate the important species differences in the intestinal respon-

ses to exogenous opiates.

The central nervous system is also a site of action for exoge-

nous opiates to affect intestinal motility. An early experiment

showed that methadone altered intestinal motility through a vagally

mediated mechanism (Scott et al., 1947) which led to the proposal that

the central nervous system was the site of action for morphine-induced

constipation (Vaughan Williams, 1954). Subsequently, intracerebral

administration of morphine to mice was shown to produce a greater

inhibition of intestinal propulsion than did subcutaneous administra-

tion (Margolin, 1954; Green, 1959). It was later suggested that this

effect could be humorally mediated (Plekss and Margolin, 1968;

Margolin, 1963) as spinal cord section or vagotomy did not block the

antipropulsive effects of intracerebally-administered morphine.

The central nervous system as a site of action for opiate-

induced constipation has been confirmed in subsequent studies in the

rat (Parolaro et al., 1977; Stewart et al., 1978; Schulz et al.,

1979), cat (Stewart et al., 1977) and dog (Bueno and Fioramonti,

1982). Despite this considerable volume of supporting evidence the

results of other studies indiciate that the central nervous system

does not have a role in opiate-induced constipation and that a direct

local action on the intestine is responsible (Tavani et al., ,1980).

Thus, the relative contributions of central and peripheral mechanisms

to the antipropulsive effects of exogenously-administered opiates

remains a point of some controversy.

The effects of exogenous opiates on gastrointestinal motility

have assumed increased interest and importance following the iden-

tification of stereospecific opiate binding sites or receptors in the

gastrointestinal tract and central nervous system (Pert and Snyder,

1973; Simon et al., 1973) and the isolation and characterization of

15

16

several endogenous peptides with potent opiate-like activity (Hug~es et

al., 1975; Li and Chung, 1976; Cox et al., 1976; Goldstein et al.,

1979). It has recently become apparent that there are three distinct

classes of opioid peptides; the endorphins, the enkephalins and the

dynorphin-related peptides (Cox, 1982). The endorphins, including e-

endorphin, are found largely in the pituitary and hypothalamus (Bloom

et al., 1978) and are derived from a larger precursor, proopiomelano-

cortin (Mains et al., 1977). The enkephalins are the second class of

opioid peptide and are widely distributed in the brain, spinal cord

and peripheral tissues, including the gastrointestinal tract (Hughes et

al., 1977; Schultzberg et al., 1978; Miller and Pickel, 1980).

Although methionine enkephalin is the N-terminal pentapeptide of e-

endorphin, the distribution (Stengaard-Pedersen and Larsson, 1981) and

biosynthetic pathways of these peptides differ markedly. Methionine

enkephalin, and in smaller quantities leucine enkephalin, are derived

from proenkephalin A (Kakidani et al., 1982). The dynorphin-related

peptides, including a-neoendorphin and to a lesser extent leucine

enkephalin, are derived from proenkephalin B (Kakidani et al., 1982).

These peptides are also widely'distributed in the brain, pituitary and

peripheral tissues (Goldstein et al., 1979; Tachibana et al., 1982).

Each class of opioid peptides has been shown to inhibit the

electrically-induced contractions of the guinea-pig ileum (Cox et al.,

1976; Hughes et al., 1975; Goldstein et al., 1979) and the presence of

the enkephalins and dynorphin in intestinal nerves suggests that these

peptides participate in the regulation of intestinal motility. There

is also some indirect functional evidence supporting a role for these

peptides in control of intestinal contractions. Dynorphin levels have

been shown to increase in the bathing medium during fatigue of the

peristaltic reflex in vitro, while incubating the preparation with

naloxone increases the frequency of peristatltic waves (Kromer and

pretzlaff, 1979; Kromer, 1980).

17

The effects of opioid peptides on intestinal contractions that

have been described here have been found in isolated tissue prepara-

tions only and very little is known about the actions of these peptides

on intestinal motility in the intact animal. Gillan and Pollock (1981)

have found that, in the rat, morphine, methionine and leucine enkepha-

lins can inhibit colonic contractions produced by stimulating the

motor efferents of the spinal cord but would also produce contractions

of the unstimulated colon. In these studies the opioids were given

systemically and no conclusions could be made as to the site of

action. A previous study (Cowan et al., 1976) had demonstrated an

inhibition of intestinal transit following intracerebroventricular

administration to mice of methionine and leucine enkephalin. However,

an antipropulsive effect was seen only with very high doses and again

it would be difficult to make firm conclusions concerning the site of

action. These peptides are also unstable in vivo and the high doses

may have been required to overcome rapid degradation of the peptides.

Since that time a number of stabilized enkephalin analogs have been

synthesized which possess a longer biological half-life in vivo. One

such analog has been shown to inhibit intestinal transit in the rat

following central administration (Schulz et al., 1979) but there is

still no information concerning the effects of the other classes of

opioid peptides or their analogs on intestinal transit. Another point

to consider when discussing the opioid peptides is the existence of

several subclasses of opioid receptor. At least three distinct types

of opioid receptor have been identified using pharmacological (Martin

et al., 1976; Gilbert et al., 1976; Lord et al., 1977) and biochemical

(Chang et al., 1979; Chang and Cuatrecasas, 1979) techniques. The

responses mediated at each type of receptor are unknown at this time

and the biological effects produced by the three classes of opioid

peptides may differ based on their relative affinity for each receptor

subclass.

The Irritable Bowel Syndrome

18

Opioid peptide control of intestinal motility at both local and

central sites may be an important topic for both basic and clinical

research. There are a number of motility disorders, including the

irritable bowel syndrome, that appear to be related to the emotional

state of the individual. In addition, the symptoms of this disorder

are exacerbated by emotional or psychological stress, a clear

illustration of the central nervous system producing changes in

gastrointestinal motility. The irritable bowel syndrome is the most

common disorder of bowel motility seen in medical clinics in the

United States and Great Britain (Almy, 1957; Ruoff, 1973). The irri-

table bowel syndrome (IBS) is a collection of symptoms which include

abdominal cramps, diarrhea, constipation or diarrhea alternating with

constipation. The diagnosis of IBS is generally made by exclusion of

all other possible diseases and a careful patient history (Kirsner,

1981). In addition to the gastrointestinal problems, IBS patients are

generally found to be very anxious individuals who score high on a

variety of psychological tests for emotional disorders (Young et al.,

1976; Whitehead et al., 1980; Latimer et al., 1981).

19

The relationship of emotional state to colonic motility had

been established in early studies of IBS patients. These patients

demonstrated a marked increase in motility and spastic contractions of

the sigmoid colon during a discussion of emotionally charged topics

(Almy et al., 1949). Similar changes could be produced in healthy

individuals by discussion of emotion provoking topics or by producing

experimental stress (Almy and Tubin, 1947; Almy et al., 1949). In

addition to this experimental evidence for emotional influences on

colonic motility, many IBS patients report an onset or worsening of

their symptoms during stressful periods (Chaurdonay and Truelove, 1962;

Young et al., 1976). More recent studies of colonic motility in IBS

patients have shown a marked alteration in contractile and myoelectric

activity. Snape and coworkers (1977) have reported an increase in the

frequency of 3 cycle/minute contractions and colonic slow wave activity

in IBS subjects when compared to controls. Subsequent studies have

confirmed this observation that a slower contractile frequency predomi-

nates in IBS patients (Whitehead et al., 1980; Latimer et al., 1981).

Motility of the small intestine has not been as extensivley studied in

relationship to IBS due to the difficulty of using endoscopic pro-

ceedures for examining the small intestine without first sedating the

patient. The use of a sedative in this situation poses a problem due

to the relationship of intestinal motility to the emotional state of

the subject.

20

Although blood levels of several gut hormones known to

influence gastrointestinal motility, including gastrin, neurotensin and

motilin, are unchanged in IBS patients, the role of other neuropeptides

in this disorder has not been investigated. There are several

conflicting reports concerning the efficacy of naloxone treatment for

the symptoms of irritable bowel (Ambinder et al., 1980; Fielding and

O'Malley, 1979), however, there is a considerable amount of circumstan-

tial evidence suggesting that the opioid peptides may at least par-

ticipate in this symptom complex.

a-Endorphin has been proposed to function as a neuroendocrine

peptide released into the circulation along with ACTH during stressful

situations (Guillemin et al., 1977) and a-endorphin release is under

the same neurochemical control as ACTH (Vale et al., 1981). In addi-

tion, methionine and leucine enkephalin and several C-terminally

extended enkephalins with opioid activity have been identified in the

adrenal medulla (Lewis et al., 1980) and these peptides are released

concomittantly with catecholamines following cholinergic stimulation

(Viveros et al., 1979). No target tissue has been established for

these circulating endorphins or enkephalins but it is possible that

gastrointestinal function may be affected by these circulating peptides

or biologically active fragments whose levels can rise during stress.

Enkephalinergic or endorphinergic containing neurons within the

brain may also participate in the regulation of autonomic outflow from

the CNS to the gut. Endorphinergic neurons originating within the

21

hypothalamus project to several brainstem regions including the reticu-

lar formation, periaqueductal gray and locus ceruleus (Childers, 1980).

The periaqueductal grey has shown to be a possible site for morphine's

intestinal antipropulsive effects (Sala et al., 1983). Enkephalin

containing neurons have also been located· in the anterior hypothalamus

which also contains a high density of opiate binding sites. In addi-

tion, the amygdyla sends enkephalin-containing processes to the stria

terminalis and its nulcei (Uhl et al., 1978) and contains the highest

density of opiate receptors in the brain (Simantov et al., 1976).

These are important observations as early work on CNS control of

gastrointestinal motility has shown that electrical stimulation of the

anterior hypothalamus enhances gastric (Fennegan and Puiggari, 1965),

small intestinal and colonic motility (Wang et al., 1940) while

electrical stimualtion of the amygdyla inhibits gastric motility

(Fennegan and Puiggari, 1965). Autoradiographic studies of opiate

receptor distribution in the medulla have revealed high densities of

opiate binding sites in the solitary nuclei, nucleus ambiguus, dorsal

motor nucleus of the vagus and on the vagus nerve itself (Atweh and

Kuhar, 1977) suggesting that the endogenous opioid peptides can modu-

late afferent input from the viscera as well as efferent outflow to a

number of visceral structures including the gut.

The data described above indicate that the endorphins and

enkephalins may be important neuromodulators of autonomic outflow to

the gastrointestinal tract and of the emotional state of the individual

as indicated by the high density of opiate receptors on limbic struc-

22

tures such as the amygdyla. It is in both of these areas that the

symptoms of the irritable bowel syndrome arise.

Statement of the Problem

Previous studies have shown that morphine could alter gastroin-

testinal motility by an action within the central nervous system. The

present study was designed to provide further support for the

centrally-mediated effect by comparing the relative potencies for inhi-

bition of intestinal transit by morphine given by several routes of

administration. While much of the work concerni.ng the site of

morphine's action on gut motility has been done in the rat, very little

is known about the contractile state of the intestine following

morphine treatment. A system was developed for direct measurement of

intestinal contractions in the unanesthetized rat before and after

morphine treatment.

The effect of endogenous opioid peptides on intestinal transit

in the rat was also unknown and intestinal transit was evaluated in

rats treated intracerebroventricularly and peripherally with several

opioid peptides as a means of determining a site of action. The

• • cerebrally-mediated effects of opioids on intestinal motility suggests

the existence of an opioid sensitive mechanism in the brain that when

stimulated can alter gut motility. Several physiological and pharma-

cological stimuli were used in an attempt to activate this system with

the intent of developing an animal model for the irritable bowel

syndrome. Finally, the possibility that a single class of opioid

receptor is mediating the intestinal effects of centrally-administered

opioids was investigated using several opioid agonists which were

highly selective for a single class of receptor.

23

METHODS

Surgical Preparation of Animals for Intestinal Transit Studies

In all experiments male or female Sprague-Dawley rats were used

and the preparation was similar for each type of experiment. These

techniques were a modification of the procedures developed by Poulakos

and Kent (1973) for intraluminal instillation of non-absorbable

radioactive markers. In some experiments, only small intestinal can-

nulas were implanted while in others intragastric, small intestinal and

large intestinal cannuals were implanted. In each case, silastic can-

nulas were used. The small intestinal cannula consisted of a 20 cm

long piece of silastic tubing (Dow Corning, Midland, MI; 0.02 in. I.D.

x 0.037 in. O.D.) with a small bulb of silicone rubber (General

Electric RTV-112, Waterford, N.Y.) fixed 2 em from the intestinal end.

The intestinal end of the cannula was sealed with a small plug of

petroleum jelly to prevent efflux of the intestinal contents.

Each rat was anesthetized with ketamine HCI (Ketalar, Parke

Davis, Detroit MI) 100 mg/kg given intraperitoneally and the proximal

small intestine was exposed through a midline abdominal incision. The

intestinal end of the cannula was pulled through a cutaneous puncture

in the midlumbar region of the animal's back and was brought sub-

cutaneously to the abdominal incision. A small stab wound was made in

the abdominal wall and the cannula was pulled into the abdominal

cavity. The tip of the cannula was introduced into the intestinal

lumen through a small incision (approximately 2 cm from the pyloric

24

25

region) and was fastened in place by tying a suture (4-0 silk) around

the silicone bulb. The abdominal incision was closed with a single set

of 4-0 silk sutures that passed through the abdominal musculature and

the skin. A second suture was used to close the cutaneous puncture and

to secure the exposed end of the intestinal cannula. The cannula was

then coiled under a gauze sponge and was kept in place by a masking

tape harness. Implantation of the intragastric and colonic cannulas

was essentially identical to this procedure with only a few modifica-

tions. The intragastric cannula was of the same inside and outside

diameter except the length was greater than 30 cm. This longer cannula

distinguished it from the small intestinal cannula in animals that had

been implanted with both intestinal and intragastric cannulas. The

intragastric cannula was implanted in the fundic region of the stomach

and was secured by passing a suture through the stomach wall and tying

it around the silicone bulb.

The colonic cannula was also made of silastic tubing but of a

larger diameter (0.025 in I.D. x 0.04 in O.D.) and was approxiamtely 20

cm in length. The larger diameter cannula distinguished it from the

small intestinal and intragastric cannulas in animals that had been

implanted with each type of cannula. The larger diameter also per-

mitted instillation of a more viscous marker used for measurement of

colonic transit as will be described in more detail. The colonic can-

nula was also fitted with a small silicone bulb approximately 0.5 cm

from the intestinal end which had been sealed with petroleum jelly.

The cannula was inserted into the colonic lumen through an incision

26

approximately 1 em from the colonic-cecal junction and was secured in a

manner similar to that used for the small intestinal cannula.

Intracerebroventricular Cannulas

Direct administration of drugs into the cerebral ventricles

required prior implantation of a ventricular cannula. The method used

in these studies was a modification of the procedure developed by

Robison et al. (1969). Polyethylene tubing (PE-10, Clay Adams,

Parsipanny, N.Y.) was passed through a small metal coil of a device

designed to pass electrical current through the coil to generate heat.

When heated, a small expansion of the tubing was produced inside the

coil. The tubing was removed and was cut on one end 4 mm from the base

of the raised portion and 5 em from this area on the other end. The

cannula was inserted (under ketamine anesthesia) into the right lateral

cerebral ventricle (4.0 mm below the skull surface) through a small

hole drilled in the skull surface. The hole was drilled with a hand-

held pin vise 2.0 mm lateral and 2.0 mm posterior to bregma. A second

hole was drilled 2.0 mm anterior and lateral to bregma and a small

stainless steel screw (J. I. Morris Co. Framingham, MA) was inserted.

The cannula was secured to the skull with a small mound of dental acry-

lic (Codesco Supply, Tucson, AZ) and the head wound was closed with

wound clips. The cannula was filled with 5.0 ~l of saline to flush out

any blood or cerebrospinal fluid and the tip was sealed closed with a

heated forceps.

Hypophysectomy

Hypophysectomized rats and aged matched, sham-operated controls

were purchased from Taconic Farms Animal Breeders (Taconic, N. Y.).

Upon arrival at the local animal facility the operated rats were pro-

vided with drinking water containing 5.0% glucose and 1.0% NaCI. All

animals were allowed to stay in their cages for 3-4 days prior to

implantation of small intestinal and intracerebroventricular cannulas.

Spinal Cord Section

27

Some of the motor efferents from the central nervous system

leave the spinal cord as the splanchnic nerves and in the rat they

emerge from the cord below thoracic vertabrae number 4. This input to

the gut was eliminated by severing the spinal cord between thoracic

vertabrae 2 and 3. This was accomplished under an operating microscope

using a number 11 scapel blade. Sham-operated animals had their spinal

cord exposed but not severed. Following spinal cord section small

intestinal and i.c.v. cannulas were implanted as described previously

and each rat was placed in a cage which rested on a heating pad. This

procedure helped to maintain body temperature during the two day reco-

very period between surgery and the experiment. Also during the these

two days each rat was fed twice daily with 5.0 m1 of a 5% glucose solu-

tion via a feeding needle.

Subdiaphramatic Vagotomy

Elimination of the vagal input to the intestine was performed in

rats that had been prepared two days previously with small intestinal

28

and i.c.v. cannulas. On the morning of the experiment each rat was

anesthetized lightly with ether and the esophagus was ,exposed by

removing the sutures that had closed the midline abdominal incision.

Two sutures were placed around the esophagus, one close to the diaphram

and the second just proximal to the esophageal-gastric junction. The

sutures were pulled tight and the esophagus was severed. In addition,

all surrounding connective tissue was also cut. Sham-operated animals

had the sutures placed only loosely around the esophagus. These ani-

mals were allowed to recover from this procedure for two hours prior to

initiation of the experiment.

Evaluation of Intestinal Transit and Gastric Emptying

Gastric Emptying

Changes in gastric in response to drug or other treatment were

evaluated by instilling approximately 0.6 ~Ci of [3H]-polyethylene gly-

col 900 (New England Nuclear, Boston, MA) 0.5m1 saline into the gastric

lumen via the implanted cannula. Thirty five minutes after instilla-

tion of the non-absorbable marker the rat was killed by cervical dislo-

cation and the stomach was removed. The stomach was placed into a

large centrifuge tube and brought to a final volume of 20 m1 with nor-

mal saline. A standard sample was prepared by adding 0.5 m1 of the

tritiated marker to 19.5 m1 of saline. The stomach samples and the

standard were homogenized (Tekmar) and centrifuged (15 minutes, 6800 x

g). A 200 ~l aliquot of the supernatant of each tube was added to 5 ml

of scintillation cocktail (Aquamix, Westchem, Tucson, Az). and each

sample was counted for 5 miuntes (Beckman L8-250, 1.0% error). The

29

number of disintegrations per minute in each stomach sample was divided

by the number of disintegrations per minute in the standard sample to

determine the percentage of administered marker that remained in each

stomach. Percent gastric emptying was calculated by subtracting from

100 the percentage of administered marker remaining in each sample.

Small and Large Intestinal Transit

Approximately 0.5 ~Ci of radiochromium as Na5lCr04 (New England

Nuclear, Boston, ~~) in 0.2 ml of saline was instilled into the duode-

num via the previously implanted cannula. The marker for large bowel

transit consisted of Na5lCr04 saline/5.0% xanthum gum which producad

a marker that was similar in consistency to normal colonic contents.

This marker was instilled into the large bowel via the cannula (0.5

~Ci, 0.2 ml volume). Twenty five or thirty five minutes after marker

instillation the rats were killed by cervical dislocation and the small

and large intestines were excised. The small and large intestines were

each divided into ten equal segments on a ruled template. The intesti-

nal segments were placed consecutively into culture tubes and the

amount of radioactivity in each segment was determined by gamma

counting (Tracor Analytic. Elk Grove IL). The amount of radioactivity

in each small or large intestinal segment was then expressed as a frac-

tion of the total radiaoctivity that was found in the small intestine

or large intestine. Intestinal transit was then quantitated by calcu-

lating the geometric center of the distribution of radioactive marker

in the small or large intestine using the following formula:

Geometric Center = ~(fraction of counts in segment X segment no.)

30

The geometric center is the center of gravity of the distribution of

marker in the intestine and can range from a value of 1.0 where all the

marker is in the first intestinal segment to 10.0 with all the marker

in the last intestinal segment. Treatments which inhibit intestinal

transit decrease the value of the geometric center while treatments

which stimulate intestinal transit increase the value of the geometric

center. This technique has proven to be a reliable measure of drug-

induced changes in intestinal transit and it is sensitive to changes in

both the distribution and leading edge of the marker (Miller et al.,

1981). As there is a maximum inhibition of transit as indicated by a

geometric center of 1.0, calculation of the EDSO value for drugs

affecting intestinal transit was greatly simplified. The EDSO is that

dose of drug which produces a half-maximal inhibition of intestinal

transit. This value is calculated from a linear regression on the

dose-response curve for percent maximum inhibition of intestinal tran-

sit produced by each dose of drug. Percent maximum inhibition is

calculated as follows:

% Maximum inhibition of Transit = (drug-control/1.0-control) X 100

where drug is the geometric center of each drug treated animal and

control is the mean geometric center of the control for each experiment

and 1.0 is the maximum inhibitIon of intestinal transit. This calcula-

tion allows a direct comparison of potencies for a drug given by dif-

ferent routes of administration or between different drugs given by the

same route.

Effects of Morphine on Small Intest~aal Transit and Gastric Emptying.

31

Female rats were prepared with small intestinal and intragastric

cannulas as previously described. Intracerbroventricular cannulas were

also implanted in some rats. All animals were placed in individual

cages and allowed to recover for 72 hours. These experiments were done

in rats that had been fasted 18 hours prior to the experiment.

Morphine sulfate dissolved in saline was administered subcutaneously

(1.0 ml/kg volume) and i.c.v. (5.0 ~l) 20 minutes prior to marker while

intragastric morphine (2.0 ml/kg volume) was given 30 minutes prior to

marker instillation. Thirty five minutes after the intragastric and

intestinal markers had been instilled the rats were killed and gastric

emptying and intestinal transit were determined. Naloxone (2 mg/kg

s.Cw) antagonism of the intestinal effects of morphine was investigated

in animals that had been implanted with intestinal cannulas only.

Differences between treated and control groups were assessed

using Dunnett's t-test for comparing several groups to a single control

and Student's t-test for grouped data.

Direct Measurement of Small Intestinal Motility

A simple and inexpensive technique for measurement of small

intestinal motility in the unanestheitzed rat was developed. An

implant conSisting of silastic tubing, 23-gauge hypodermic needles, a 6

cc syringe and dental acrylic was used for these studies. A small

length (12 cm) of silas tic tubing was fixed to a 23 gauge hypodermic

needle cut to 0.5 cm in length. The connection was sealed with sili-

cone rubber. Two of these tubing-needle combinations were used for

32

each implant and were fixed in a small rubber mold. The plunger was

removed from a 6 cc plastic syringe and the top 2 cm of the barrel was

used as a support matrix inserted into the rubber mold. Dental acrylic

was placed into the mold, allowed to set and the implant was removed

from the mold. A small bulb of silicone rubber was fixed approximately

1 cm from the intestinal end of the cannulas (see figure 1). The

implant was soaked in 70% ethanol before implantation. Rats were

anesthetized with ketamine and a midline abdominal incision as well as

a small incision between the shoulders were made. The cannulas were

brought subcutaneously from the shoulders into the abdominal cavity

through a puncture in the abdominal wall. The dental acrylic plug was

secured between the shoulders using a purse string suture. The

intestinal ends of each cannula were inserted through small incisions

in the proximal duodenum and proximal jejunum and were secured by

fastening a silk suture around the silicone bulb. Most of the radioac-

tive marker in the intestinal transit studies was in the proximal 50 %

of the intestine (see figure 2). In order to correlate changes in

transit with alterations in intestinal contractions, motility was

recorded from the proximal portion of the small intestine. Animals

fitted with recording cannulas were housed and fasted on the same sche-

dule as that used in the intestinal transit experiments. On the day of

the experiment, rats were placed in a plastic restrainer for 30 minutes

after which the cannulas were connected, via the 23 gauge needles, to

an infusion pump (Harvard Apparatus) and a pressure transducer (Statham

P23Db) using a three-way stopcock. The cannulas were perfused at a

23 gao Needles

I 6 cc Syringe Silastic

Tubing

RTV 112 Silicone Rubber

Figure 1. Schematic drawing of the implant used to record intestinal motility from unanesthetized rats.

33

34

rate of 0.04 ml/min with distilled water and motility was recorded as

pressure increases resulting from changes in outflow resistance as the

intestine contracted. Pressure tracings were recorded on a Beckman 511

Dynograph (9853A coupler, 30 Hz high frequency cut-off). The rate of

pressure increase in this system when the lumen of a cannula was

abruptly occluded was 2.9 cm of H20 per second. The fall in pressure

was more rapid with a rate of 12.6 cm of H20 per second. A control

recording was obtained for 30 minutes, efter which morphine was admi-

nistered either s.c. or i.c.v. and intestinal contractions were

recorded for an additional 60 minutes. The records were analyzed by

visual inspection and the number of contractions occurring in both

areas of the small intestine was recorded. Changes in the frequency of

contractions were expressed as a percentage of that occurring in the

thirty minute control period for each rat. Data were analyzed by

Student's t-test for paired data.

Effects of Opioid Peptides on Intestinal Transit in Castor Oil Treated Rats

Si1astic small intestinal cannulas were implanted into the

duodenum of female rats and some animals were also prepared with i.c.v.

cannulas. All animals were housed individually for 48 hours and were

fasted for 18 hours prior to the start of the experiment. These

experiments were initiated by instilling 0.5 ml of castor oil (Fisher

Scientific Products, Tustin, CA) into the duodenum. Thirty minutes

later the opioid peptides, a-endorphin and [D-a1a2-methionine5]enkeph-

a1inamide (Beckman Bioproducts, Palo Alto, CA) were given either intra-

cerebroventricu1a1ry (i.c.v.) or intraperitonea1ly (i.p.), while the

peptides dynorphin (1-13) and [D-ala2-leucineS]enkephalinamide

(Peninsula Laboratories, San Carlos, CA) were given i .. c.v. Thirty

minutes after the peptides were administered radiochromium was

instilled into the intestine and after an additional 25 minutes the

rats were killed·and small intestinal transit in each rat was deter-

mined. A separate group of animals was used to determine the anti-

diarrheal effects of the opioid peptides. Small intestinal weight and

percent body weight loss were determined following castor oil and pep-

tide treatment. The animals were treated using the same protocol

(without radiochromium instillation) used to evaluate intestinal tran-

sit. Differences in body weight loss and intestinal transit between

groups were assessed using analysis of variance and Student's t-test

for grouped data.

Effects of Electroconvulsive Shock on Gastrointestinal Motility and Analgesia

35

Intragastric, small and large intestinal cannulas were implanted

in male Sprague-Dawley rats (280-340 g, Division of Animal Resources,

University of Arizona). The animals were housed individually and

allowed to recover for 72 hours and were fasted 18 hours prior to the

experiment. The rats were pretreated with saline or naloxone (1.0

mg/kg s.c.) followed after 10 minutes by transocular electroconvulsive

shock (ECS, 150 rnA, 0.5 sec duration) or sham-ECS. Five minutes after

ECS the radioactive markers were instilled into the gastrointestinal

tract. The animals were killed thirty-five minutes later and gastric

emptying, small intestinal and large intestinal transit were evaluated.

A separate group of animals was used to determine whether ECS treat-

ment could produce naloxone-reversible analgesia as had been reported

previously (Lewis et al., 1981; Holaday and Belenky, 1980). Thermal

analgesia was determined using a 52°C hot-plate test. Groups of 6-9

rats were pretreated with saline or naloxone (1.0 or 5.0 mg/kg s.c.)

followed after 10 minutes by ECS or sham-ECS. Analgesia was tested at

5 and 35 minutes after ECS. The time to rear-paw lick or an escape

attempt from the plexiglass box that surrounded the hot-plate surface

was timed and a 60 second maximum cut-off time was used. Response

latencies were converted to percent maximum possible effect (% M.P.E.)

using the following formula:

% M.P.E.·= (Test Latency-Control Latency/60-Control Latency) X 100

where test latency is the time to rear-paw lick or an escape attemtpt

following ECS treatment, control is the pretreatment latency obtained

for each rat and 60 is the maximum time each rat could remain on the

hot-plate. Data were analyzed by analysis of variance and Student's t-

test for grouped data.

Effects of Inescapable Footshock on Gastrointestinal Motility and Analgesia

Intragastric, small intest~nal and large intestinal cannulas

were implanted in the gastrointestinal tract of male Sprague-Dawley

rats (280-340 g, Division of Animal Resources, University of Arizona).

Each animal was housed individually and allowed to recover for 72 hours

and fasted 18 hours prior to the experiment. Each rat was then placed

in a plexiglass box with a grid floor and a shock scrambler was used to

apply electrical current (3.75 mA, 1 shock/5 sec) to the grid floor for

20 minutes.

37

Sham treated animals were placed in the box for 20 minutes with no

current applied to the floor. Immediately following the cessation of

shock or sham treatment the radioactive markers were instilled into

the gastrointestinal tract. After an additional 35 minutes, the rats

were killed and small intestinal and large intestinal transit and

gastric emptying were evaluated. Previous studies (Akil et al.,1976;

Watkins et sl., 1980) have shown that inescapable footshock (IFS) could

produce a naloxone-reversible analgesia. The analgesic effects of IFS

were determined in animals that had been pretreated with saline or

naloxone (10 mg/kg s.c.). Twenty minutes after pretreatment, rats were

placed in the plexiglass box for either shock or sham treatment for

twenty minutes. Immediately following and at 10 minutes after removal

from the shock box, the rats were placed on the 52 0C hot-plate and the

latency to rear-paw lick or an escape attempt was timed. Percent

M.P.E. was calculated as described previously. Data were analyzed by

analysis of variance and Student's t-test for grouped data.

Kyotorphin Effects on Intestinal Transit and Analgesia

Kyotorphin is a dipeptide first isolated from bovine brain and

produces opioid effects by promoting enkephalin release from enkephali-

nergic neurons (Takagi et al., 1979). Silastic small intestinal can-

nulas and polyethylene i.c.v. cannulas were implanted into female

Sprague-Dawley rats (200-240 g, Division of Animal Resources,

University of Arizona). The rats were housed individually for 72 hours

and were fasted 18 hours prior to the experiment. Kyotorphin

38

(Peninsula Laboratories, San Carlos, CA) was given i.c.v. and ten minu-

tes later radiochromium was instilled into the duodenum. After an

additional 35 minutes the rats were killed and small intestinal transit

was evaluated.

The analgesic effects of kyotorphin were determined in a

separate group of animals in which only i.c.v. cannulas had been

implanted. Rats were pretreated with naloxone (2.0 or 5.0 mg/kg s.c.)

or saline followed after 10 minutes by i.c.v. kyotorphin (15, 30, 60 or

120 ~g). The analgesic effects of kyotorphin were determined at 10,

20, and 40 minutes post-peptide treatment on the 52 0C hot-plate.

Percent maximum possible effect was calculated as described previously

and data were analyzed by Dunnett's t-test and Student's t-test for

grouped data.

Determination of Opioid Receptor Selectivity of Agonists In Vitro

The opioid agonists examined in these studies included nor-

morphine, a-endorphin, [D-Ala2 , Met5]enkephalinamide (DALA), [D-ala2 ,

MePhe4 , Gly-oI5]enkephalin (DAGO), cyclic [D-pen2 , L-Cys5]enkephalin

(DPLCE), cyclic [D-Pen2 , L-pen5]enk~phalin (DPLPE), [D-Pen2 ,

D-Pen5]enkephalin (DPDPE) and [D-Ala2 , D-Leu5]enkephalin (DADL). All

drugs except the cyclic enkephalins were obtained commercially. The

cyclic enkephalins were synthesized by solid-phase methods that have

been described elesewhere (Mosberg et al., 1983a,b). Receptor selec-

tivity of each of these agonists was estimated by comparing the poten-

cies of each compound for inhibition of the electrically-induced

contractions of the guinea-pig ileum longitudinal muscle, myenteric

39

plexus (G.P.I.) to that of the mouse vas deferens (M.V.D.). The M.V.D.

is believed to contain predominately delta type opioid receptors while

the G.P.I. contains predominately the mu type of receptor (Lord et al.,

1977). The IC50 is the concentration of agonist required to produce a

half-maximal inhibition of the contraction height and is an indicator

of affinity of a drug for its receptor. The ratio of IC50 values for

each of the compounds in the G.P.I. and M.V.D. can be used as an index

of the preference of each agonist for the receptors found in each of

the preparations. A large G.P.I./M.V.D. ratio indicates a greater

selectivity for the receptor found in the M.V.D., the delta receptor.

The vasa deferentia of male CD-I and ICR (25-35 g) mice were removed

and mounted in a tis'sue bath after the procedure developed by

~enderson et al. (1972). Briefly, the vasa were removed and stripped

of any connective tissue and blood vessels and a pair of vasa were used

for each preparation. The two tissues were tied together and were

fastened at each end to a 14 K gold chain using 5-0 silk thread. The

tissue was then connected to the bottom of the tissue bath and to a

Grass isometric force transducer (Model FT030). The tissue was bathed

in Mg++ free Krebs' bicarbonate buffer warmed to 37 °C and bubbled with

95% 02 5% C02. The preparation was stimulated transmurally (100 V,

1100 ~A, 0.1 Hz, 2.0 msec duration) using platinum electrodes and a

Grass S44D stimulator. Contractile responses were recorded on a Grass

oscillographic recorder (Model 2200S). The preparations were stimu-

lated for 30 minutes during which time the buffer in the bath was

changed several times. Agonists were added in volumes of 10-300 ~l and

40

remained in contact with the tissue for 3 minutes after which the

buffer was changed until the pre-drug twitch height was restored.

Subsequent doses were added at 15 minute intervals. The ICsO was

calculated from the regression line of the dose response curve for each

preparation. Naloxone Ke values (the dissociation constant of the

antagonist) was calculated using the single dose method of Kosterlitz

and Watt (1968). The Ke was calculated using the following formula:

Ke = a/DR-1

where a is the agonist concentration in riM and DR is the dose ratio of

ICsO values obtained in the presence and absence of naloxone.

The G.P.I. was prepared after the methods used by Kosterlitz et

ale (1970). A glass rod was inserted into the lumen of a 3 em segment

of guinea-pig (Hartley, either sex) ileum. A scapel blade was used to

make a small cut through the longitudinal muscle with attatched myen-

teric plexus along the mesenteric attatchment. The longitudinal

muscle, myenteric plexus was then separated from the rest of the ileal

segmen